Cell Renewal and Cell Death

In order to maintain a constant number of cells in adult tissues and organs, cell death must be balanced by cell
proliferation.Most tissues therefore contain cells that are able to proliferate as required to replace cells that have
died. Moreover, in some tissues a subpopulation of cells divide continuously throughout life to replace cells that
have a high rate of turnover in adult animals. Cell renewal is thus carefully regulated to maintain properly sized and
functioning adult tissues and organs.

• Most types of differentiated cells in adult animals are no longer capable of proliferation. If these cells are
lost, they are replaced by the proliferation of less differentiated cells derived from self-renewing stem cells.
• Other types of differentiated cells, however, retain the ability to proliferate as needed to repair damaged
tissue throughout the life of the organism. These cells enter the G0 stage of the cell cycle but resume
proliferation as needed to replace cells that have been injured or died.

Fibroblasts, which are dispersed in connective tissues where they secrete collagen.
Skin fibroblasts are normally arrested in G0 but rapidly proliferate if needed to repair damage resulting
from a cut or wound.
Blood clotting at the site of a wound leads to the release of platelet-derived growth factor (PDGF) from blood
platelets.
PDGF activates a receptor tyrosine kinase, stimulating both the proliferation of fibroblasts and their migration into
the wound, where their proliferation and secretion of collagen contributes to repair and regrowth of the damaged
tissue.

The endothelial cells that line blood vessels are another type of fully differentiated cell that remains capable of
proliferation. Proliferation of endothelial cells allows them to form new blood vessels as
needed for repair and regrowth of damaged tissue. Endothelial cell proliferation and the resulting formation of blood
capillaries is triggered by a growth factor (vascular endothelial growth factor, or VEGF) produced by cells of the
tissue that the new capillaries will invade. The production of VEGF is in turn triggered by a lack of oxygen, so the
result is a regulatory system in which tissues that have a low oxygen supply resulting from insufficient circulation
stimulate endothelial cell proliferation and
recruit new capillaries

Smooth muscle cells, which form the walls of larger blood vessels (e.g., arteries) as well as the contractile portions
of the digestive and respiratory tracts and other internal organs.

Epithelial cells of some internal organs are also able to proliferate to replace damaged tissue.
e.g. Liver

STEM CELLS
Subpopulation of less differentiated self-renewing cells called stem cells that are present in most adult tissues.
• As they retain the capacity to proliferate and replace differentiated cells throughout the lifetime of an
animal, stem cells play a critical role in the maintenance of most tissues and organs.
• The key property of stem cells is that they divide to produce one daughter cell that remains a stem cell and
one that divides and differentiates
• Stem cells typically give rise to rapidly proliferating cells (called transit amplifying cells) that undergo
several divisions before they differentiate, so division of a stem cell leads to the production of multiple
differentiated cells.
• Stem cells are self-renewing populations that can serve as a source for the production of differentiated cells
throughout life.

Hematopoetic cells (Cell renewal)

1. Lymphoid -------------T lymphocyte and B Lymohocyte
2. Myeloid -
a. Reticulocyte - Erythrocytes
 b. Megakaryocyte - Platelets
c. Monocyte - Macrophage
d. Granulocytes - Neutrophil, Basophil, Easinophil

Intestinal epithelium (Cell renewal)

Stem cell found in the botton of crypts
Forms ---- Absorptive cells, Goblet cells and Enetro endocrine cells

Skin (Cell renewal)

Epidermis - stem cell found in the single basal layer
Hair follicles - Found in the region called bulge
Sebaceous gland

Skeletal system (Repair damage)

Found below basal lamina and are called Satellite cells

Hematopoietic stem cell transplantation (or bone marrow transplantation)

Important role in the treatment of a variety of cancers.

Most cancers are treated by chemotherapy with drugs that kill rapidly dividing cells by damaging DNA or inhibiting
DNA replication. These drugs do not act selectively against cancer cells and so are also toxic to those normal tissues
that are dependent on continual renewal by stem cells, such as blood,
skin, hair, and the intestinal epithelium.

The hematopoietic stem cells are among the most rapidly dividing cells of the body, so the toxic effects of anticancer
drugs on these cells frequently limit the effectiveness of chemotherapy in cancer treatment.
Hematopoietic stem cell transplantation provides an approach to bypassing this toxicity, thereby allowing the use of
higher drug doses to treat the patient’s cancer more effectively.

In this procedure, the patient is treated with high doses of chemotherapy that would normally not be tolerated
because of toxic effects on the hematopoietic system.
The potentially lethal damage is repaired, however, by transferring new hematopoietic stem cells (obtained from
either bone marrow or peripheral blood) to the patient following completion of chemotherapy.
In some cases, the stem cells are obtained from the patient prior to chemotherapy, stored, and then returned to the
patient once chemotherapy is completed. However, it is important to ensure that these cells are not contaminated
with cancer cells.
Alternatively, the stem cells to be transplanted can be obtained from a healthy donor (usually a close relative) whose
tissue type closely matches the patient.

In addition to their use in cancer treatment, hematopoietic stem cell transfers are used to treat patients with diseases
of the hematopoietic system, such as hemoglobin disorders and immune deficiencies

Embryonic stem cells

While adult stem cells are difficult to isolate and culture, it is relatively straightforward to isolate and propagate the
stem cells of early embryos (embryonic stem cells).
The ability to give rise to all of the differentiated cell types of adult organisms ---Pluripotency

Embryonic stem cells are pluripotent and retain the capacity to develop into all of the different types of cells in adult
tissues and organs.In addition, they can be induced to differentiate into a variety of different types of cells in culture.

Embryonic stem cells are grown in the presence of growth factors that maintain the cells in their undifferentiated
state. If these factors are removed from the medium, the cells aggregate into structures
that resemble embryos (embryoid bodies) and then differentiate into a wide range of cell types. Moreover,
embryonic stem cells can be directed to differentiate along specific pathways by the addition of appropriate growth
factors or small molecules (e.g., retinoic acid) to the culture medium

These differentiated cells have been used for treatment of mouse models of cardiac arrhythmia,
Parkinson’s disease, and spinal cord injury.

Somatic cell nuclear transfer

Transfer of nuclei from adult somatic cells into enucleated eggs to create cloned offspring.
Only 1-2% of embryos generally give rise to offsprings and they normally have limited lifespan.

Both the inefficiency of cloning by somatic cell nuclear transfer and the abnormalities prevalent
in cloned animals are thought to reflect the difficulties in completely reprogramming the
epigenetic state of an adult nucleus, including reversal of DNA methylation.

In therapeutic cloning, a nucleus from an adult human cell would be transferred to an enucleated egg, which would
then be used to produce an early embryo in culture. Embryonic stem cells could then be cultured from the cloned
embryo and used to generate appropriate types of differentiated cells for transplantation therapy.
The major advantage provided by therapeutic cloning is that the embryonic stem cells derived by this procedure
would be genetically identical to the recipient of the transplant, who was the donor of the
adult somatic cell nucleus.
This bypasses the barrier of the immune system in rejecting the transplanted tissue.

Induced pluripotent stem cells

Conversion of adult somatic cells directly to pluripotent stem cells in culture with the help of around 4 transcription
factor combinations introduced by retroviral genetic transfer.
All of these combinations of transcription factors that induce pluripotency activate a transcriptional program that is
also expressed in embryonic cells and maintains the pluripotent state.
Three core transcription factors (Oct4, Sox2, and Nanog) play central roles in this program
These factors form an autoregulatory loop, in which they act together to positively regulate each other’s expression.
In addition, they activate expression of other genes that maintain the pluripotent state, while repressing
genes that enable differentiation along specific cell lineages.
The positive autoregulation at the core of this transcriptional program maintains pluripotency,
while allowing the cells to undergo differentiation in response to appropriate signals.

Transient expression of reprogramming factors

To establish pluripotent stem cells by transient expression of the reprogramming factors, without the need for stable
integration of viral genomes or foreign genes.
Once the recipient somatic cell is reprogrammed to the pluripotent state, autoregulation maintains pluripotency
without the need for continued expression of the factors that initially induced the reprogramming.
- Important because some transcription factor used were found to be oncogenes and also some retro viral genes were
found to be harmful to the recipient.

Transdifferentiation of somatic cells

Somatic cells reprogrammed to other types of differentiated cells—a phenomenon termed transdifferentiation.
Differentiated blood cells can also be reprogrammed either to other blood cell types or to hematopoietic stem cells,
potentially providing a new source of patient-specific cells for hematopoietic stem cell transplantation.
Transdifferentiation thus provides a direct route to generating desired types of differentiated cell populations,
bypassing the need for pluripotent stem cells and allowing the direct conversion of adult cells from a patient into
differentiated cells that could be used for transplantation therapy

Programmed Cell Death

Responsible for balancing cell proliferation and maintaining constant cell numbers in tissues undergoing cell
turnover.
Provides a defense mechanism by which damaged and potentially dangerous cells can be eliminated for the good of
the organism as a whole.
Eliminate cells carrying potentially harmful mutations, including cells with mutations that might lead to the
development of cancer.

For example,
programmed cell death is responsible
for the elimination of larval tissues during amphibian and insect metamorphosis
for the elimination of tissue between the digits during the formation of fingers and toes

Development of mammalian nervous system;

50% of developing neurons are eliminated by programmed cell death. Those that survive are selected for having
made the correct connections with their target cells, which secrete growth factors that signal cell survival by
blocking the neuronal cell death program.

Necrosis-
Accidental death of cells that results from an acute injury
Cells that die by necrosis swell and lyse, releasing their contents into the extracellular space and cause inflammation.

Apoptosis-
Programmed cell death [an active process], which usually proceeds by a distinct series of cellular changes.During
apoptosis,
a. Chromosomal DNA is usually fragmented as a result of cleavage between nucleosomes.
b. The chromatin condenses and the nucleus then breaks up into small pieces.
c. Finally, the cell itself shrinks and breaks up into membrane-enclosed fragments called apoptotic bodies.

Apoptotic cells and cell fragments are efficiently recognized and phagocytosed by both macrophages and
neighboring cells, so cells that die by apoptosis are rapidly removed from tissues.
The removal of apoptotic cells is mediated by the expression of so-called “eat me” signals on the cell surface.
These signals include phosphatidylserine, which is normally restricted to the inner leaflet of
the plasma membrane.
During apoptosis, phosphatidylserine becomes expressed on the cell surface, where it is recognized by receptors
expressed by phagocytic cells.

In 1986 mutagenesis of C. elegans identified two genes that were required for developmental cell death (ced-3 and
ced-4).
• If either ced-3 or ced-4 was inactivated by mutation, the normal programmed cell deaths did not take place.
• ced-9, functioned as a negative regulator of apoptosis. If ced-9 was inactivated by mutation, the cells that
would normally survive failed to do so.Conversely, if ced-9 was expressed at an abnormally high level, the
normal programmed cell deaths failed to occur.

It was later found that proteins encoded by these genes acted in a pathway with
Ced-4 acting to stimulate Ced-3, and Ced-9 inhibiting Ced-4

Ced-3 is the prototype of a family of more than a dozen proteases, known as caspases because they have cysteine
(C) residues at their active sites and cleave after aspartic acid (Asp) residues in their substrate proteins.
The caspases are the ultimate effectors or executioners of programmed cell death, bringing about the events of
apoptosis by cleaving more than 100 different target proteins.

One key target of the caspases is an inhibitor of a DNase, which when activated is responsible for fragmentation of
nuclear DNA.
• Caspases cleave- nuclear lamins, leading to fragmentation of the nucleus;
• Cleave cytoskeletal proteins, leading to disruption of the cytoskeleton,blebbing (irregular bulging) of the
plasma membrane, and cell fragmentation;
 • Cleave golgi matrix proteins, leading to fragmentation of the Golgi apparatus.
• Caspases also cleave and activate a “scramblase” that translocates phosphatidylserine from the inner to the
outer leaflet of the plasma membrane.

All caspases are synthesized as inactive precursors (called procaspases) that can be converted to the active form
by proteolytic cleavage, catalyzed by other caspases.
Initiator caspases are activated directly in response to the various signals that induce apoptosis.
The initiator caspases then cleave and activate the effector caspases, which are responsible for digesting the cellular
target proteins that mediate the events of apoptosis.

Ced-4 functioned as an activator of the caspase Ced-3.

Ced-4 and its mammalian homolog (Apaf-1) bind to caspases and promote their activation.
Ced-4 and Apaf-1 are in turn controlled by proteins encoded by members of a large gene family related to ced-9.

The Bcl-2 family

bcl-2, was first identified in 1985 as an oncogene that contributed to the development of human B-cell lymphomas
(cancers of B lymphocytes).
Bcl-2 was found to inhibit apoptosis.
[Cancer cells are generally defective in the normal process of programmed cell death and that their inability to
undergo apoptosis is as important as their uncontrolled proliferation in the development of malignant tumors.]

Bcl 2 family is divided into three functional groups.

Some members of the Bcl-2 family—like Bcl-2 itself—are antiapoptotic regulatory proteins that inhibit
programmed cell death.
Other family members, however, are proapoptotic proteins that act to induce caspase activation and promote cell
death.
There are two groups of these proapoptotic proteins,
the proapoptotic regulatory proteins and the proapoptotic effector proteins.

The proapoptotic effector proteins are the downstream effectors that directly induce apoptosis.

The proapoptotic regulatory proteins are upstream members of the cascade, regulated by the signals that induce cell
death (e.g., DNA damage) or cell survival (e.g., growth factors).
When activated, the proapoptotic regulatory proteins initiate apoptosis by two mechanisms:
(1) they antagonize the antiapoptotic regulatory proteins and
(2) they activate the proapoptotic effector proteins.

The mitochondrial pathway of apoptosis In mammalian cells,

Many cell death signals induce apoptosis as a result of damage to mitochondria.
When active, the proapoptotic effector proteins Bax and Bak form oligomers in the outer membrane of
mitochondria, resulting in the release of cytochrome c from the intermembrane space.
Release of cytochrome c leads to the formation of apoptosomes containing Apaf-1 and caspase-9 in which caspase-
9[initiator caspase] is activated.
Caspase-9 then activates downstream caspases, such as caspase-3[effector caspase], by proteolytic cleavage and thus
causes cell death.

Signaling pathways that regulate apoptosis

The pathways that induce apoptosis in mammalian cells are grouped as intrinsic or extrinsic pathways, which
differ in their involvement of Bcl-2 family proteins and in the identity of the caspase that initiates cell death.
The intrinsic pathway is activated by DNA damage and other forms of cell stress, whereas
the extrinsic pathway is activated by signals from other cells.

One important role of apoptosis is the elimination of damaged cells, so apoptosis is stimulated by many forms of cell
stress, including DNA damage, viral infection, and growth factor deprivation. These stimuli activate the intrinsic
pathway of apoptosis, which leads to release of cytochrome c from
mitochondria and activation of caspase-9.

Intrinsic pathways-

1.
DNA damage is one of the principal triggers of programmed cell death, leading to the elimination of cells carrying
potentially harmful mutations.
In mammalian cells, a major pathway leading to cell cycle arrest in response to DNA damage is mediated by the
transcription factor p53
The ATM and Chk2 protein kinases, which are activated by DNA damage, phosphorylate and stabilize p53. The
resulting increase in p53 leads to transcriptional activation of p53 target genes. These include the Cdk inhibitor p21,
which inhibits Cdk2/cyclin E complexes, halting cell cycle progression
in G1

However, activation of p53 by DNA damage can also lead to apoptosis.

The induction of apoptosis by p53 results, from transcriptional activation of genes encoding the proapoptotic
regulatory proteins PUMA and Noxa.
Increased expression of these proapoptotic proteins leads to activation of Bax and Bak, release of cytochrome c from
mitochondria, and activation of caspase-9.

Thus p53 mediates both cell cycle arrest and apoptosis in response to DNA damage. Whether DNA damage in a
given cell leads to apoptosis or reversible cell cycle arrest depends on the extent of damage and the resulting level of
p53 induction, as well as on the influence of other life/death signals being received by the cell.

2.
Growth factor deprivation is another form of cell stress that activates the intrinsic pathway of apoptosis. In this case,
apoptosis is controlled by signaling pathways that promote cell survival by inhibiting apoptosis in response to
growth factor stimulation.

example-
Programmed cell death in development is provided by the vertebrate nervous system. About 50% of neurons die by
apoptosis, with the survivors having received sufficient amounts of survival signals from their target cells.
These survival signals are polypeptide growth factors related to nerve growth factor (NGF), which induces both
neuronal survival and differentiation by activating a receptor tyrosine kinase.

One of the major intracellular signaling pathways responsible for promoting cell survival is initiated by the enzyme
PI 3-kinase, which is activated by either tyrosine kinases or G protein-coupled receptors.

PI 3-kinase phosphorylates the membrane phospholipid PIP2 to form PIP3, which activates the serine/
threonine kinase Akt
Akt then phosphorylates a number of proteins that regulate apoptosis

• One key substrate for Akt is the proapoptotic regulatory protein called Bad. Phosphorylation of Bad by Akt
creates a binding site for 14-3-3 chaperone proteins that sequester Bad in an inactive form, so
phosphorylation of Bad by Akt inhibits apoptosis and promotes cell survival. Bad is similarly
phosphorylated by protein kinases of other growth factor-induced signaling pathways, including the
Ras/Raf/ MEK/ERK pathway.

• Another targets of Akt, includes the FOXO transcription factors, and play key roles in cell survival.
Phosphorylation of FOXO by Akt creates a binding site for 14-3-3 proteins, which sequester FOXO in an
inactive form in the cytoplasm.In the absence of growth factor signaling and Akt activity, FOXO is released
 from 14-3-3 and translocates to the nucleus, stimulating transcription of proapoptotic genes, including the
gene encoding the proapoptotic regulatory protein, Bim.

• Akt and its downstream target GSK-3 also regulate other transcription factors with roles in cell survival,
including p53 and NF-κB, which control the expression of additional Bcl-2 family members.

• The antiapoptotic Bcl-2 family member Mcl-1 is targeted for ubiquitylation and degradation by GSK-3, and
its level is also modulated via translational regulation by both GSK-3 and the mTOR pathway.

Extrinsic Pathways-
Some secreted polypeptides activate receptors that induce cell death via the extrinsic pathway of apoptosis.
These receptors directly activate a distinct initiator caspase—caspase-8.
The polypeptides that signal cell death by this pathway belong to the tumor necrosis factor (TNF) family. They
bind to members of the TNF receptor family, which can induce apoptosis in a variety of cell types.
One of the best-characterized members of this family is the cell surface receptor Fas, which plays important roles
in controlling cell death in the immune system. For example, apoptosis induced by activation of Fas is responsible
for killing target cells of the immune system, such as cancer cells or virusinfected cells, as well as for eliminating
excess lymphocytes at the end of an immune response.

TNF and related family members consist of three identical polypeptide chains, and their binding induces receptor
trimerization.
The cytoplasmic portions of the receptors bind adaptor molecules that in turn bind caspase-8.
This leads to activation of caspase-8, which can then cleave and activate downstream effector caspases. In some
cells, caspase-8 activation and subsequent activation of caspases-3 and -7 is sufficient to induce apoptosis directly.
In other cells, however, amplification of the signal is needed. This results from caspase-8 cleavage of the
proapoptotic regulatory protein Bid, leading to Bid activation, permeabilization of mitochondria, and activation of
caspase-9, thus amplifying the caspase cascade initiated by direct activation of caspase-8 at cell death receptors.

Alternative pathways of programmed cell death

Autophagy
Autophagy provides a mechanism for the gradual turnover of the cell’s components by the uptake of proteins or
organelles into vesicles (autophagosomes) that fuse with lysosomes.
One role of autophagy is to promote cell survival under conditions of nutrient deprivation.
Activation of autophagy under these conditions leads to increased degradation of cellular proteins and other
macromolecules, allowing their components to be degraded as a source of energy or reutilized
for essential functions.
In some circumstances, however, autophagy can lead to cell death by degrading essential cell components.

In mammals, autophagy may contribute to cell death induced by a variety of pathological treatments, such as drug
treatments or infection with some viruses.
Autophagic cell death does not require caspases and, rather than possessing the distinct morphological features of
apoptosis, the dying cells are characterized by an accumulation of lysosomes.

Regulated necrotic cell death (necroptosis)

induced as a programmed response to stimuli such as infection or DNA damage. Under these conditions, necroptosis
provides an alternative pathway of cell death if apoptosis does not occur